CHITIN LINKS IN THE YEAST CELL WALL. SUMMARY. Work on the linkages of chitin to other yeast cell wall components was completed, establishing that chitin laid down in a ring at the mother-daughter cell neck is mostly attached to beta(1-3)glucan, whereas the chitin dispersed in the cell wall is linked to beta(1-6)glucan. The primary septum chitin is mostly free. Further work showed that the CRH1 and CRH2 genes are essential for the attachment of chitin to beta(1-6)glucan. In budding yeast, chitin is a minor but essential component of the cell wall, required for viability. Chitin is present in three locations in the cell: as a ring laid down at the base of an emerging bud, as a disk forming the primary septum at cytokinesis and as a randomly distributed polysaccharide appearing in the daughter cell wall after septation. The synthesis of the chitin at the primary septum is catalyzed by chitin synthase II, that at the other locations by chitin synthase III. Earlier work of our laboratory showed that part of the chitin is free, whereas other portions are bound either to beta(1-3)glucan or to beta(1-6)glucan, two components of the cell wall. Our finding of a specific function for the chitin ring formed at budding, i.e. preventing cell wall growth at the neck region between mother and daughter cell, suggested that the different locations of chitin may also correspond to linkages with different polysaccharides. To check on this hypothesis, I developed in previous periods a new procedure for the quantitative study of chitin bonds to individual cell wall components. Briefly, the new approach consists in labeling chitin specifically in vivo with [14-C]glucosamine, followed by preparation of cell walls and removal of proteins with alkali. The cell walls are either left untreated or digested with beta(1-3)- or beta(1-6)-glucanases to liberate the chitin attached to either one of the corresponding polysaccharides. Then, both the untreated and the enzyme-treated cell walls are completely solubilized in water by carboxymethylation, followed by size-exclusion chromatography. By analysis and comparison of elution profiles it is possible to calculate what percentage of the chitin is free or linked either to beta(1-3)- or beta(1-6)glucan. By coupling this new technique with cell synchronization and deposition of chitin at different points of the cell cycle, I could confirm our hypothesis that most of the bound chitin at the neck is attached to beta(1-3)glucan, whereas that dispersed in the cell wall is linked to beta(1-6)glucan. The chitin of the primary septum is prevalently free. This work was completed in the first part of the present period and a paper was published in the Journal of Biological Chemistry (chosen as ?Paper of the Week? by the editors). The next step was to find out how the chitin becomes attached to one or the other of the glucans. A group at the Complutense University of Madrid, Spain, headed by Javier Arroyo, had studied in recent years two genes, CRH1 and CRH2 (for Congo Red Hypersensitive), whose sequences have homology with known transglycosylases. The Congo Red hypersensitivity of crh1 and crh2 mutants suggested a relationship with cell wall structure. The distribution of Crh1p and Crh2p in the cell and its changes during the cell cycle coincided with those of at least part of the chitin. Thus, there were indications that they may be involved in chitin metabolism. I obtained mutants in both genes from Dr. Arroyo and studied the chitin distribution in them with the new procedure. The results showed that crh2 mutants were severely defective in chitin bound to beta(1-6)glucan, whereas a double crh1 crh2 mutant completely lacked chitin linked to this polysaccharide. These finding led to the beginning of an ongoing collaboration with the Arroyo group. Later results in both laboratories implicated the two proteins in the general response of the cell to stress conditions. This regulatory function is now under study. Crh1p and Crh2p are the first proteins for which an involvement in the formation of cross-links in the fungal cell wall has been demonstrated.